RESUMO
O-GlcNAc transferase (OGT) is the sole enzyme responsible for the post-translational modification of O-GlcNAc on thousands of target nucleocytoplasmic proteins. To date, nine variants of OGT that segregate with OGT Congenital Disorder of Glycosylation (OGT-CDG) have been reported and characterized. Numerous additional variants have been associated with OGT-CDG, some of which are currently undergoing investigation. This disorder primarily presents with global developmental delay and intellectual disability (ID), alongside other variable neurological features and subtle facial dysmorphisms in patients. Several hypotheses aim to explain the etiology of OGT-CDG, with a prominent hypothesis attributing the pathophysiology of OGT-CDG to mutations segregating with this disorder disrupting the OGT interactome. The OGT interactome consists of thousands of proteins, including substrates as well as interactors that require noncatalytic functions of OGT. A key aim in the field is to identify which interactors and substrates contribute to the primarily neural-specific phenotype of OGT-CDG. In this review, we will discuss the heterogenous phenotypic features of OGT-CDG seen clinically, the variable biochemical effects of mutations associated with OGT-CDG, and the use of animal models to understand this disorder. Furthermore, we will discuss how previously identified OGT interactors causal for ID provide mechanistic targets for investigation that could explain the dysregulated gene expression seen in OGT-CDG models. Identifying shared or unique altered pathways impacted in OGT-CDG patients will provide a better understanding of the disorder as well as potential therapeutic targets.
RESUMO
Pathogenic variants in the O-GlcNAc transferase gene (OGT) have been associated with a congenital disorder of glycosylation (OGT-CDG), presenting with intellectual disability which may be of neuroectodermal origin. To test the hypothesis that pathology is linked to defects in differentiation during early embryogenesis, we developed an OGT-CDG induced pluripotent stem cell line together with isogenic control generated by CRISPR/Cas9 gene-editing. Although the OGT-CDG variant leads to a significant decrease in OGT and O-GlcNAcase protein levels, there were no changes in differentiation potential or stemness. However, differentiation into ectoderm resulted in significant differences in O-GlcNAc homeostasis. Further differentiation to neuronal stem cells revealed differences in morphology between patient and control lines, accompanied by disruption of the O-GlcNAc pathway. This suggests a critical role for O-GlcNAcylation in early neuroectoderm architecture, with robust compensatory mechanisms in the earliest stages of stem cell differentiation.
Assuntos
Diferenciação Celular , Células-Tronco Pluripotentes Induzidas , Deficiência Intelectual , N-Acetilglucosaminiltransferases , Placa Neural , Fenótipo , Humanos , N-Acetilglucosaminiltransferases/genética , N-Acetilglucosaminiltransferases/metabolismo , Deficiência Intelectual/genética , Deficiência Intelectual/patologia , Células-Tronco Pluripotentes Induzidas/metabolismo , Células-Tronco Pluripotentes Induzidas/patologia , Placa Neural/metabolismo , Defeitos Congênitos da Glicosilação/genética , Defeitos Congênitos da Glicosilação/patologia , Defeitos Congênitos da Glicosilação/metabolismo , Sistemas CRISPR-Cas , Glicosilação , Edição de Genes , Células-Tronco Neurais/metabolismo , Células-Tronco Neurais/patologiaRESUMO
O-GlcNAcylation is a dynamic post-translational modification that diversifies the proteome. Its dysregulation is associated with neurological disorders that impair cognitive function, and yet identification of phenotype-relevant candidate substrates in a brain-region specific manner remains unfeasible. By combining an O-GlcNAc binding activity derived from Clostridium perfringens OGA (CpOGA) with TurboID proximity labeling in Drosophila, we developed an O-GlcNAcylation profiling tool that translates O-GlcNAc modification into biotin conjugation for tissue-specific candidate substrates enrichment. We mapped the O-GlcNAc interactome in major brain regions of Drosophila and found that components of the translational machinery, particularly ribosomal subunits, were abundantly O-GlcNAcylated in the mushroom body of Drosophila brain. Hypo-O-GlcNAcylation induced by ectopic expression of active CpOGA in the mushroom body decreased local translational activity, leading to olfactory learning deficits that could be rescued by dMyc overexpression-induced increase of protein synthesis. Our study provides a useful tool for future dissection of tissue-specific functions of O-GlcNAcylation in Drosophila, and suggests a possibility that O-GlcNAcylation impacts cognitive function via regulating regional translational activity in the brain.
Newly synthesized proteins often receive further chemical modifications that change their structure and role in the cell. O-GlcNAcylation, for instance, consists in a certain type of sugar molecule being added onto dedicated protein segments. It is required for the central nervous system to develop and work properly; in fact, several neurological disorders such as Alzheimer's, Parkinson's or Huntington's disease are linked to disruptions in O-GlcNAcylation. However, scientists are currently lacking approaches that would allow them to reliably identify which proteins require O-GlcNAcylation in specific regions of the brain to ensure proper cognitive health. To address this gap, Yu et al. developed a profiling tool that allowed them to probe O-GlcNAcylation protein targets in different tissues of fruit flies. Their approach relies on genetically manipulating the animals so that a certain brain area overproduces two enzymes that work in tandem; the first binds specifically to O-GlcNAcylated proteins, which allows the second to add a small 'biotin' tag to them. Tagged proteins can then be captured and identified. Using this tool helped Yu et al. map out which proteins go through O-GlcNAcylation in various brain regions. This revealed, for example, that in the mushroom body the 'learning center' of the fly brain O-GlcNAcylation occurred predominantly in the protein-building machinery. To investigate the role of O-GlcNAcylation in protein synthesis and learning, Yu et al. used an approach that allowed them to decrease the levels of O-GlcNAcylation in the mushroom body. This resulted in reduced local protein production and the flies performing poorly in olfactory learning tasks. However, artificially increasing protein synthesis reversed these deficits. Overall, the work by Yu et al. provides a useful tool for studying the tissue-specific effects of O-GlcNAcylation in fruit flies, and its role in learning. Further studies should explore how this process may be linked to cognitive function by altering protein synthesis in the brain.
Assuntos
Drosophila , Corpos Pedunculados , Animais , Encéfalo , Cognição , Processamento de Proteína Pós-TraducionalRESUMO
The addition of O-linked ß-N-acetylglucosamine (O-GlcNAc) to proteins (referred to as O-GlcNAcylation) is a modification that is crucial for vertebrate development. O-GlcNAcylation is catalyzed by O-GlcNAc transferase (OGT) and reversed by O-GlcNAcase (OGA). Missense variants of OGT have recently been shown to segregate with an X-linked syndromic form of intellectual disability, OGT-linked congenital disorder of glycosylation (OGT-CDG). Although the existence of OGT-CDG suggests that O-GlcNAcylation is crucial for neurodevelopment and/or cognitive function, the underlying pathophysiologic mechanisms remain unknown. Here we report a mouse line that carries a catalytically impaired OGT-CDG variant. These mice show altered O-GlcNAc homeostasis with decreased global O-GlcNAcylation and reduced levels of OGT and OGA in the brain. Phenotypic characterization of the mice revealed lower body weight associated with reduced body fat mass, short stature and microcephaly. This mouse model will serve as an important tool to study genotype-phenotype correlations in OGT-CDG in vivo and for the development of possible treatment avenues for this disorder.
Assuntos
Modelos Animais de Doenças , Deficiência Intelectual , N-Acetilglucosaminiltransferases , Animais , N-Acetilglucosaminiltransferases/metabolismo , N-Acetilglucosaminiltransferases/genética , N-Acetilglucosaminiltransferases/deficiência , Deficiência Intelectual/genética , Encéfalo/patologia , Encéfalo/metabolismo , Fenótipo , Camundongos , Transtornos do Neurodesenvolvimento/patologia , Transtornos do Neurodesenvolvimento/genética , Transtornos do Neurodesenvolvimento/enzimologia , beta-N-Acetil-Hexosaminidases/metabolismo , Glicosilação , Peso CorporalRESUMO
The soil bacterium Bacillus subtilis is a model organism to investigate the formation of biofilms, the predominant form of microbial life. The secreted protein BslA self-assembles at the surface of the biofilm to give the B. subtilis biofilm its characteristic hydrophobicity. To understand the mechanism of BslA self-assembly at interfaces, here we built a molecular model based on the previous BslA crystal structure and the crystal structure of the BslA paralogue YweA that we determined. Our analysis revealed two conserved protein-protein interaction interfaces supporting BslA self-assembly into an infinite 2-dimensional lattice that fits previously determined transmission microscopy images. Molecular dynamics simulations and in vitro protein assays further support our model of BslA elastic film formation, while mutagenesis experiments highlight the importance of the identified interactions for biofilm structure. Based on this knowledge, YweA was engineered to form more stable elastic films and rescue biofilm structure in bslA deficient strains. These findings shed light on protein film assembly and will inform the development of BslA technologies which range from surface coatings to emulsions in fast-moving consumer goods.
Assuntos
Proteínas de Bactérias , Matriz Extracelular de Substâncias Poliméricas , Proteínas de Bactérias/metabolismo , Matriz Extracelular de Substâncias Poliméricas/metabolismo , Biofilmes , Bacillus subtilis/metabolismo , Simulação de Dinâmica MolecularRESUMO
Protein O-GlcNAcylation is an evolutionary conserved post-translational modification catalysed by the nucleocytoplasmic O-GlcNAc transferase (OGT) and reversed by O-GlcNAcase (OGA). How site-specific O-GlcNAcylation modulates a diverse range of cellular processes is largely unknown. A limiting factor in studying this is the lack of accessible techniques capable of producing homogeneously O-GlcNAcylated proteins, in high yield, for in vitro studies. Here, we exploit the tolerance of OGT for cysteine instead of serine, combined with a co-expressed OGA to achieve site-specific, highly homogeneous mono-glycosylation. Applying this to DDX3X, TAB1, and CK2α, we demonstrate that near-homogeneous mono-S-GlcNAcylation of these proteins promotes DDX3X and CK2α solubility and enables production of mono-S-GlcNAcylated TAB1 crystals, albeit with limited diffraction. Taken together, this work provides a new approach for functional dissection of protein O-GlcNAcylation.
Assuntos
Processamento de Proteína Pós-Traducional , Proteínas , Proteínas/metabolismo , Glicosilação , N-Acetilglucosaminiltransferases/genética , N-Acetilglucosaminiltransferases/metabolismo , Acetilglucosaminidase/metabolismo , Acetilglucosamina/metabolismoRESUMO
Protein O-GlcNAcylation is a monosaccharide post-translational modification maintained by two evolutionarily conserved enzymes, O-GlcNAc transferase (OGT) and O-GlcNAcase (OGA). Mutations in human OGT have recently been associated with neurodevelopmental disorders, although the mechanisms linking O-GlcNAc homeostasis to neurodevelopment are not understood. Here, we investigate the effects of perturbing protein O-GlcNAcylation using transgenic Drosophila lines that overexpress a highly active OGA. We reveal that temporal reduction of protein O-GlcNAcylation in early embryos leads to reduced brain size and olfactory learning in adult Drosophila. Downregulation of O-GlcNAcylation induced by the exogenous OGA activity promotes nuclear foci formation of Polycomb-group protein Polyhomeotic and the accumulation of excess K27 trimethylation of histone H3 (H3K27me3) at the mid-blastula transition. These changes interfere with the zygotic expression of several neurodevelopmental genes, particularly shortgastrulation (sog), a component of an evolutionarily conserved sog-Decapentaplegic (Dpp) signaling system required for neuroectoderm specification. Our findings highlight the importance of early embryonic O-GlcNAcylation homeostasis for the fidelity of facultative heterochromatin redeployment and initial cell fate commitment of neuronal lineages, suggesting a possible mechanism underpinning OGT-associated intellectual disability.
Assuntos
Drosophila , Heterocromatina , Animais , Humanos , Drosophila/genética , Drosophila/metabolismo , Heterocromatina/genética , Processamento de Proteína Pós-Traducional , Homeostase , Desenvolvimento Embrionário/genética , N-Acetilglucosaminiltransferases/genética , N-Acetilglucosaminiltransferases/metabolismoRESUMO
O-linked ß-N-acetylglucosamine (O-GlcNAc) transferase (OGT) is an essential enzyme that modifies proteins with O-GlcNAc. Inborn OGT genetic variants were recently shown to mediate a novel type of congenital disorder of glycosylation (OGT-CDG), which is characterised by X-linked intellectual disability (XLID) and developmental delay. Here, we report an OGTC921Y variant that co-segregates with XLID and epileptic seizures, and results in loss of catalytic activity. Colonies formed by mouse embryonic stem cells carrying OGTC921Y showed decreased levels of protein O-GlcNAcylation accompanied by decreased levels of Oct4 (encoded by Pou5f1), Sox2 and extracellular alkaline phosphatase (ALP), implying reduced self-renewal capacity. These data establish a link between OGT-CDG and embryonic stem cell self-renewal, providing a foundation for examining the developmental aetiology of this syndrome.
Assuntos
Deficiência Intelectual , Animais , Camundongos , Deficiência Intelectual/metabolismo , Autorrenovação Celular , Glicosilação , N-Acetilglucosaminiltransferases/genética , N-Acetilglucosaminiltransferases/metabolismoRESUMO
O-GlcNAcylation is a dynamic post-translational modification performed by two opposing enzymes: O-GlcNAc transferase and O-GlcNAcase. O-GlcNAcylation is generally believed to act as a metabolic integrator in numerous signalling pathways. The stoichiometry of this modification is tightly controlled throughout all stages of development, with both hypo/hyper O-GlcNAcylation resulting in broad defects. In this Primer, we discuss the role of O-GlcNAcylation in developmental processes from stem cell maintenance and differentiation to cell and tissue morphogenesis.
Assuntos
Processamento de Proteína Pós-Traducional , Células-Tronco , Glicosilação , Células-Tronco/metabolismo , Transdução de Sinais , N-Acetilglucosaminiltransferases/metabolismo , Diferenciação CelularRESUMO
Life-threatening hyperammonemia occurs in both inherited and acquired liver diseases affecting ureagenesis, the main pathway for detoxification of neurotoxic ammonia in mammals. Protein O-GlcNAcylation is a reversible and nutrient-sensitive post-translational modification using as substrate UDP-GlcNAc, the end-product of hexosamine biosynthesis pathway. Here we show that increased liver UDP-GlcNAc during hyperammonemia increases protein O-GlcNAcylation and enhances ureagenesis. Mechanistically, O-GlcNAcylation on specific threonine residues increased the catalytic efficiency for ammonia of carbamoyl phosphate synthetase 1 (CPS1), the rate-limiting enzyme in ureagenesis. Pharmacological inhibition of O-GlcNAcase, the enzyme removing O-GlcNAc from proteins, resulted in clinically relevant reductions of systemic ammonia in both genetic (hypomorphic mouse model of propionic acidemia) and acquired (thioacetamide-induced acute liver failure) mouse models of liver diseases. In conclusion, by fine-tuned control of ammonia entry into ureagenesis, hepatic O-GlcNAcylation of CPS1 increases ammonia detoxification and is a novel target for therapy of hyperammonemia in both genetic and acquired diseases.
Assuntos
Amônia , Carbamoil-Fosfato Sintase (Amônia) , Hiperamonemia , Ureia , Difosfato de Uridina , Acetilglucosamina , Amônia/metabolismo , Animais , Biocatálise , Carbamoil-Fosfato Sintase (Amônia)/genética , Carbamoil-Fosfato Sintase (Amônia)/metabolismo , Modelos Animais de Doenças , Glicosilação , Humanos , Hiperamonemia/genética , Hiperamonemia/metabolismo , Mamíferos/metabolismo , Camundongos , N-Acetilglucosaminiltransferases/genética , N-Acetilglucosaminiltransferases/metabolismo , Acidemia Propiônica/genética , Acidemia Propiônica/metabolismo , Processamento de Proteína Pós-Traducional/genética , Ureia/metabolismo , Difosfato de Uridina/genética , Difosfato de Uridina/metabolismoRESUMO
Aspergillus fumigatus is a devastating opportunistic fungal pathogen causing hundreds of thousands of deaths every year. Phosphoglucose isomerase (PGI) is a glycolytic enzyme that converts glucose-6-phosphate to fructose-6-phosphate, a key precursor of fungal cell wall biosynthesis. Here, we demonstrate that the growth of A. fumigatus is repressed by the deletion of pgi, which can be rescued by glucose and fructose supplementation in a 1:10 ratio. Even under these optimized growth conditions, the Δpgi mutant exhibits severe cell wall defects, retarded development, and attenuated virulence in Caenorhabditis elegans and Galleria mellonella infection models. To facilitate exploitation of A. fumigatus PGI as an antifungal target, we determined its crystal structure, revealing potential avenues for developing inhibitors, which could potentially be used as adjunctive therapy in combination with other systemic antifungals. IMPORTANCE Aspergillus fumigatus is an opportunistic fungal pathogen causing deadly infections in immunocompromised patients. Enzymes essential for fungal survival and cell wall biosynthesis are considered potential drug targets against A. fumigatus. PGI catalyzes the second step of the glycolysis pathway, linking glycolysis and the pentose phosphate pathway. As such, PGI has been widely considered as a target for metabolic regulation and therefore a therapeutic target against hypoxia-related diseases. Our study here reveals that PGI is important for A. fumigatus survival and exhibit pleiotropic functions, including development, cell wall glucan biosynthesis, and virulence. We also solved the crystal structure of PGI, thus providing the genetic and structural groundwork for the exploitation of PGI as a potential antifungal target.
Assuntos
Aspergillus fumigatus , Glucose-6-Fosfato Isomerase , Antifúngicos/farmacologia , Aspergillus fumigatus/metabolismo , Parede Celular/metabolismo , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Glucose-6-Fosfato Isomerase/genética , Glucose-6-Fosfato Isomerase/metabolismo , Humanos , VirulênciaRESUMO
Protein O-GlcNAcylation is a dynamic posttranslational modification that is catalyzed by the enzyme O-GlcNAc transferase (OGT) and is essential for neurodevelopment and postnatal neuronal function. Missense mutations in OGT segregate with a novel X-linked intellectual disability syndrome, the OGT congenital disorder of glycosylation (OGT-CDG). One hypothesis for the etiology of OGT-CDG is that loss of OGT activity leads to hypo-O-GlcNAcylation of as yet unidentified, specific neuronal proteins, affecting essential embryonic, and postnatal neurodevelopmental processes; however, the identity of these O-GlcNAcylated proteins is not known. Here, we used bioinformatic techniques to integrate sequence conservation, structural data, clinical data, and the available literature to identify 22 candidate proteins that convey OGT-CDG. We found using gene ontology and PANTHER database data that these candidate proteins are involved in diverse processes including Ras/MAPK signaling, translational repression, cytoskeletal dynamics, and chromatin remodeling. We also identify pathogenic missense variants at O-GlcNAcylation sites that segregate with intellectual disability. This work establishes a preliminary platform for the mechanistic dissection of the links between protein O-GlcNAcylation and neurodevelopment in OGT-CDG.
Assuntos
Deficiência Intelectual Ligada ao Cromossomo X , N-Acetilglucosaminiltransferases , Processamento de Proteína Pós-Traducional , Biologia Computacional , Deficiências do Desenvolvimento/genética , Glicosilação , Humanos , Deficiência Intelectual Ligada ao Cromossomo X/genética , Mutação de Sentido Incorreto , N-Acetilglucosaminiltransferases/genética , Processamento de Proteína Pós-Traducional/genética , SíndromeRESUMO
O-GlcNAcylation is a reversible co-/post-translational modification involved in a multitude of cellular processes. The addition and removal of the O-GlcNAc modification is controlled by two conserved enzymes, O-GlcNAc transferase (OGT) and O-GlcNAc hydrolase (OGA). Mutations in OGT have recently been discovered to cause a novel Congenital Disorder of Glycosylation (OGT-CDG) that is characterized by intellectual disability. The mechanisms by which OGT-CDG mutations affect cognition remain unclear. We manipulated O-GlcNAc transferase and O-GlcNAc hydrolase activity in Drosophila and demonstrate an important role of O-GlcNAcylation in habituation learning and synaptic development at the larval neuromuscular junction. Introduction of patient-specific missense mutations into Drosophila O-GlcNAc transferase using CRISPR/Cas9 gene editing leads to deficits in locomotor function and habituation learning. The habituation deficit can be corrected by blocking O-GlcNAc hydrolysis, indicating that OGT-CDG mutations affect cognition-relevant habituation via reduced protein O-GlcNAcylation. This study establishes a critical role for O-GlcNAc cycling and disrupted O-GlcNAc transferase activity in cognitive dysfunction, and suggests that blocking O-GlcNAc hydrolysis is a potential strategy to treat OGT-CDG.
Assuntos
Drosophila , Deficiência Intelectual , Acetilglucosamina/genética , Acetilglucosamina/metabolismo , Animais , Drosophila/genética , Drosophila/metabolismo , Habituação Psicofisiológica/genética , Humanos , Hidrolases/genética , Deficiência Intelectual/genética , N-Acetilglucosaminiltransferases/genética , N-Acetilglucosaminiltransferases/metabolismo , Processamento de Proteína Pós-Traducional/genéticaRESUMO
Aspergillus fumigatus is the causative agent of invasive aspergillosis, an infection with mortality rates of up to 50%. The glucan-rich cell wall of A. fumigatus is a protective structure that is absent from human cells and is a potential target for antifungal treatments. Glucan is synthesized from the donor uridine diphosphate glucose, with the conversion of glucose-6-phosphate to glucose-1-phosphate by the enzyme phosphoglucomutase (PGM) representing a key step in its biosynthesis. Here, we explore the possibility of selectively targeting A. fumigatus PGM (AfPGM) as an antifungal treatment strategy. Using a promoter replacement strategy, we constructed a conditional pgm mutant and revealed that pgm is required for A. fumigatus growth and cell wall integrity. In addition, using a fragment screen, we identified the thiol-reactive compound isothiazolone fragment of PGM as targeting a cysteine residue not conserved in the human ortholog. Furthermore, through scaffold exploration, we synthesized a para-aryl derivative (ISFP10) and demonstrated that it inhibits AfPGM with an IC50 of 2 µM and exhibits 50-fold selectivity over the human enzyme. Taken together, our data provide genetic validation of PGM as a therapeutic target and suggest new avenues for inhibiting AfPGM using covalent inhibitors that could serve as tools for chemical validation.
Assuntos
Aspergilose , Aspergillus fumigatus , Antifúngicos/farmacologia , Aspergilose/tratamento farmacológico , Aspergilose/microbiologia , Aspergillus fumigatus/enzimologia , Aspergillus fumigatus/genética , Glucanos/metabolismo , Humanos , Fosfoglucomutase/genética , Fosfoglucomutase/metabolismoRESUMO
Aspergillus fumigatus is a human opportunistic pathogen showing emerging resistance against a limited repertoire of antifungal agents available. The GTPase Rho1 has been identified as an important regulator of the cell wall integrity signaling pathway that regulates the composition of the cell wall, a structure that is unique to fungi and serves as a target for antifungal compounds. Rom2, the guanine nucleotide exchange factor to Rho1, contains a C-terminal citron homology (CNH) domain of unknown function that is found in many other eukaryotic genes. Here, we show that the Rom2 CNH domain interacts directly with Rho1 to modulate ß-glucan and chitin synthesis. We report the structure of the Rom2 CNH domain, revealing that it adopts a seven-bladed ß-propeller fold containing three unusual loops. A model of the Rho1-Rom2 CNH complex suggests that the Rom2 CNH domain interacts with the Rho1 Switch II motif. This work uncovers the role of the Rom2 CNH domain as a scaffold for Rho1 signaling in fungal cell wall biosynthesis.
Assuntos
Aspergillus fumigatus/metabolismo , Fatores de Transcrição de Zíper de Leucina Básica/metabolismo , Parede Celular/fisiologia , Proteínas Fúngicas/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas rho de Ligação ao GTP/metabolismo , Aspergillus fumigatus/genética , Aspergillus fumigatus/crescimento & desenvolvimento , Fatores de Transcrição de Zíper de Leucina Básica/química , Fatores de Transcrição de Zíper de Leucina Básica/genética , Proteínas Fúngicas/química , Proteínas Fúngicas/genética , Peptídeos e Proteínas de Sinalização Intracelular/química , Peptídeos e Proteínas de Sinalização Intracelular/genética , Conformação Proteica , Proteínas Serina-Treonina Quinases/química , Proteínas Serina-Treonina Quinases/genética , Transdução de Sinais , Proteínas rho de Ligação ao GTP/química , Proteínas rho de Ligação ao GTP/genéticaRESUMO
O-GlcNAcylation is an essential post-translational modification that has been implicated in neurodevelopmental and neurodegenerative disorders. O-GlcNAcase (OGA), the sole enzyme catalyzing the removal of O-GlcNAc from proteins, has emerged as a potential drug target. OGA consists of an N-terminal OGA catalytic domain and a C-terminal pseudo histone acetyltransferase (HAT) domain with unknown function. To investigate phenotypes specific to loss of OGA catalytic activity and dissect the role of the HAT domain, we generated a constitutive knock-in mouse line, carrying a mutation of a catalytic aspartic acid to alanine. These mice showed perinatal lethality and abnormal embryonic growth with skewed Mendelian ratios after day E18.5. We observed tissue-specific changes in O-GlcNAc homeostasis regulation to compensate for loss of OGA activity. Using X-ray microcomputed tomography on late gestation embryos, we identified defects in the kidney, brain, liver, and stomach. Taken together, our data suggest that developmental defects during gestation may arise upon prolonged OGA inhibition specifically because of loss of OGA catalytic activity and independent of the function of the HAT domain.
Assuntos
Desenvolvimento Embrionário/fisiologia , beta-N-Acetil-Hexosaminidases/metabolismo , Animais , Domínio Catalítico , Desenvolvimento Embrionário/genética , Feminino , Histona Acetiltransferases/metabolismo , Histona Acetiltransferases/fisiologia , Homeostase , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , N-Acetilglucosaminiltransferases/metabolismo , Gravidez , Processamento de Proteína Pós-Traducional , Microtomografia por Raio-X/métodos , beta-N-Acetil-Hexosaminidases/genética , beta-N-Acetil-Hexosaminidases/fisiologiaRESUMO
Aspergillus fumigatus is an opportunistic mold responsible for severe life-threatening fungal infections in immunocompromised patients. The cell wall, an essential structure composed of glucan, chitin, and galactomannan, is considered to be a target for the development of antifungal drugs. The nucleotide sugar donor GDP-mannose (GDP-Man) is required for the biosynthesis of galactomannan, glycosylphosphatidylinositol (GPI) anchors, glycolipid, and protein glycosylation. Starting from fructose-6-phosphate, GDP-Man is produced by the sequential action of the enzymes phosphomannose isomerase, phosphomannomutase (Pmm), and GDP-mannose pyrophosphorylase. Here, using heterokaryon rescue and gene knockdown approaches we demonstrate that the phosphomannomutase encoding gene in A. fumigatus (pmmA) is essential for survival. Reduced expression of pmmA is associated with significant morphological defects including retarded germination, growth, reduced conidiation, and abnormal polarity. Moreover, the knockdown strain exhibited an altered cell wall organization and sensitivity toward cell wall perturbing agents. By solving the first crystal structure of A. fumigatus phosphomannomutase (AfPmmA) we identified non-conservative substitutions near the active site when compared to the human orthologues. Taken together, this work provides a genetic and structural foundation for the exploitation of AfPmmA as a potential antifungal target.
Assuntos
Aspergillus fumigatus/genética , Guanosina Difosfato Manose/metabolismo , Fosfotransferases (Fosfomutases)/genética , Fosfotransferases (Fosfomutases)/metabolismo , Antifúngicos/farmacologia , Aspergilose/tratamento farmacológico , Aspergilose/patologia , Aspergillus fumigatus/efeitos dos fármacos , Aspergillus fumigatus/metabolismo , Parede Celular/metabolismo , Deleção de Genes , Humanos , Virulência/genéticaRESUMO
O-GlcNAcylation is a post-translational modification catalysed by O-GlcNAc transferase (OGT). Missense mutations in OGT have been associated with developmental disorders, OGT-linked congenital disorder of glycosylation (OGT-CDG), which are characterized by intellectual disability. OGT relies on the hexosamine biosynthetic pathway (HBP) for provision of its UDP-GlcNAc donor. We considered whether mutations in UDP-N-acetylhexosamine pyrophosphorylase (UAP1), which catalyses the final step in the HBP, would phenocopy OGT-CDG mutations. A de novo mutation in UAP1 (NM_001324114:c.G685A:p.A229T) was reported in a patient with intellectual disability. We show that this mutation is pathogenic and decreases the stability and activity of the UAP1 isoform AGX1 in vitro. X-ray crystallography reveals a structural shift proximal to the mutation, leading to a conformational change of the N-terminal domain. These data suggest that the UAP1A229T missense mutation could be a contributory factor to the patient phenotype.
Assuntos
Deficiências do Desenvolvimento/genética , Galactosiltransferases/genética , Hexosaminas/biossíntese , Mutação de Sentido Incorreto , Sequência de Aminoácidos , Animais , Cristalografia por Raios X , Estabilidade Enzimática , Galactosiltransferases/química , Galactosiltransferases/metabolismo , Humanos , Processamento de Proteína Pós-Traducional , Homologia de Sequência de AminoácidosRESUMO
O-GlcNAcylation is an abundant and dynamic protein posttranslational modification (PTM), with crucial roles in metazoans. Studies of this modification are hampered by the lack of convenient methods for detecting native O-GlcNAcylation. Here, we describe a novel gel-based approach, Separation of O-GlcNAcylated Proteins by Polyacrylamide Gel Electrophoresis (SOPAGE), which enables detection of O-GlcNAc levels and dynamics.